1. Field of the Invention
The invention is directed to a non-hygroscopic stable crystalline form of N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-xcex2-cyclohexyl-alanine amide of formula I. The compound has antithrombotic activity, 
including the inhibition of platelet aggregation and thrombus formation in mammals, and is useful in the prevention and treatment of thrombosis associated with disease states such as myocardial infarction, stroke, peripheral arterial disease and disseminated intravascular coagulation.
In addition, the invention is directed to processes for preparing the crystalline form of N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-xcex2-cyclohexylalanine amide, a pharmaceutical composition thereof and intermediates thereof.
Haemostasis, the biochemistry of blood coagulation, is an extremely complex phenomena whereby normal whole blood and body tissue spontaneously arrest bleeding from injured blood vessels. Effective haemostasis requires the combined activity of vascular, platelet and plasma factors as well as a controlling mechanism to prevent excessive clotting. Defects, deficiencies, or excesses of any of these components can lead to hemorrhagic or thrombotic consequences.
Platelet adhesion, spreading and aggregation on extracellular matrices are central events in thrombus formation. These events are mediated by a family of adhesive glycoproteins, i.e., fibrinogen, fibronectin, and von Willebrand factor. Fibrinogen is a co-factor for platelet aggregation, while fibronectin supports platelet attachments and spreading reactions, and von Willebrand factor is important in platelet attachment to and spreading on subendothelial matrices. The binding sites for fibrinogen, fibronectin and von Willebrand factor have been located on the platelet membrane protein complex known as glycoprotein IIb/IIIa.
Adhesive glycoproteins, like fibrinogen, do not bind with normal resting platelets. However, when a platelet is activated with an agonist such as thrombin or adenosine diphosphate, the platelet changes its shape, perhaps making the GPIIb/IIIa binding site accessible to fibrinogen. The compound within the scope of the present invention blocks the fibrinogen receptor, and thus has the aforesaid antithrombotic activity.
2. Reported Developments
It has been observed that the presence of Arg-Gly-Asp (RGD) is necessary in fibrinogen, fibronectin and von Willebrand factor for their interaction with the cell surface receptor (Ruoslahti E., Pierschbacher, Cell 1986, 44, 517-18). Two other amino acid sequences also seem to take part in the platelet attachment function of fibrinogen, namely, the, Gly-Pro-Arg sequence, and the dodecapeptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val sequence (SEQ ID NO: 1). Small synthetic peptides containing the RGD or dodecapeptide have been shown to bind to the platelet GPIIb/IIIa receptor and competitively inhibit binding of fibrinogen, fibronectin and von Willebrand factor as well as inhibit aggregation of activated platelets (Plow, et al., Proc. Natl. Acad. Sci. USA 1985, 82, 8057-61; Ruggeri, et al., Proc. Natl. Acad. Sci. USA 1986, 5708-12;
Ginsberg, et al., J. Bio. Chem. 1985, 260, 3931-36; and Gartner, et al., J. Biol. Chem. 1987, 260, 11,891-94).
Indolyl compounds containing guanidinoalkanoyl- and guandinoalkenoyl-aspartyl moieties are reported to be platelet-aggregation inhibitors by Tjoeng, et al., U.S. Pat. Nos. 5,037,808 and 4,879,313.
U.S. Pat. No. 4,992,463 (Tjoeng, et al.), issued Feb. 12, 1991, discloses generically that a series of aryl and aralkyl guanidinoalkyl peptide mimetic compounds exhibit platelet aggregation inhibiting activity and discloses specifically a series of mono- and dimethoxy phenyl peptide mimetic compounds and a biphenylalkyl peptide mimetic compound.
U.S. Pat. No. 4,857,508 (Adams, et al.), issued Aug. 15, 1989, discloses generically that a series of guandinoalkyl peptide derivatives containing terminal aralkyl substituents exhibit platelet aggregation inhibiting activity and discloses specifically a series of O-methyl tyrosine, biphenyl, and naphthyl derivatives containing a terminal amide functionality.
Haverstick, D. M. et al., in Blood 66 (4), 946-952 (1985), disclose that a number of synthetic peptides, including arg-gly-asp-ser (SEQ ID NO:2) and gly-arg-gly-asp-ser (SEQ ID NO:3), are capable of inhibiting thrombin-induced platelet aggregation.
Plow, E. F. et al. in Proc. Nati. Acad Sci. USA 79, 3711-3715 (1982), disclose that the tetrapeptide glycyl-L-prolyl-L-arginyl-L-proline (SEQ ID NO:4) inhibits fibrinogen binding to human platelets.
French Application No. 86/17507, filed Dec. 15, 1986, discloses that tetra-, penta- and hexapeptide derivatives containing the xe2x80x94arg-gly-asp-sequence are useful as antithrombotics.
U.S. Pat. No. 4,683,291 (Zimmerman, et al.), issued Jul. 28, 1987, discloses that a series of peptides, comprised of from six to forty amino acids, which contain the sequence xe2x80x94arg-gly-aspxe2x80x94 are platelet binding inhibitors.
European Application Publication No. 0 319 506, published Jun. 7, 1989, discloses that a series of tetra-, penta-, and hexapeptide derivatives containing the xe2x80x94arg-gly-asp-sequence are platelet aggregation inhibitors.
Cyclic peptide analogues containing the moiety Gly-Asp are reported to be fibrinogen receptor antagonists in U.S. Pat. No. 5,023,233.
Peptides and pseudopeptides containing amino-, guanidino-, imidizaloyl, and/or amidinoalkanoyl, and alkenoyl moieties are reported to be antithrombotic agents in pending U.S. application Ser. Nos. 07/677,006, 07/534,385, and 07/460,777 filed on Mar. 28, 1991, Jun. 7, 1990, and Jan. 4, 1990, respectively, now abandoned, as well as in U.S. Pat. No. 4,952,562, and in International Application No. PCT/US90/05448, filed Sep. 25, 1990, all assigned to the same assignee as the present invention.
Peptides and pseudopeptides containing amino- and guanidino- alkyl- and alkenyl-benzoyl, phenylalkanoyl, and phenylalkenoyl moieties are reported to be antithrombotic agents in pending U.S. application Ser. No. 07/475.043, filed Feb. 5, 1990, and in International Application No. PCT/US91/02471, filed Apr. 11, 1991, published as International Publication No. WO 92/13117 Oct. 29, 1992, assigned to the same assignee as the present invention.
Alkanoyl and substituted alkanoyl azacycloalkylformyl aspartic acid derivatives are reported to be platelet aggregation inhibitors in U.S. Pat. No. 5,053,392, filed Dec. 1, 1989, and assigned to the same assignee and having the same inventorship as the present invention.
N-subsituted azacycloalkylcarbonyl cyclic aminoacylaspartic acid derivatives are reported to be antithrombotics in U.S. Pat. No. 5,064,814, filed Apr. 5, 1990 by the same inventors and assigned to the same assignee as the present invention. Azacycloalkylformylglycyl aspartic acid derivatives are reported to be antithrombotics in U.S. Pat. No. 5,051,405, filed Oct. 10, 1989, and assigned to the same assignee as the present invention.
European Patent Application 0 479 481, published Apr. 8, 1992, discloses azacycloalkyalkanoyl glycyl aspartyl amino acids as fibrinogen receptor antagonists.
European Patent Application 0 478 362, published Apr. 1, 1992, discloses azacycloalkyalkanoyl peptidyl xcex2-alanines as fibrinogen receptor antagonists.
PCT Patent Application Publication No. WO95/10295 discloses azacycloalkylalkanoyl peptides and pseudopeptides of formula II and, in 
particular, N-[N-[N-(4-(piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-xcex2-cyclohexylaianine amide that inhibit platelet aggregation and thrombus formation in mammals and are useful in the prevention and treatment of thrombosis. The N-[N-[N-(4-(piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-xcex2-cyclohexylalanine amide prepared according to PCT Patent Application Publication No. WO95/10295 is amorphous, hygroscopic and is physically unstable as it absorbs moisture. PCT Patent Application Publication No. WO95/10295 does not disclose a non-hygroscopic stable crystalline form of N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-xcex2-cyclohexyl-alanine amide.
PCT Patent Application Publication No. WO95/10295 also discloses that the azacycloalkylalkanoyl peptides and pseudopeptides are prepared generally by standard solid phase or solution phase peptide synthesis procedures using starting materials and/or readily available intermediates from chemical supply companies such as Aldrich or Sigma, (H. Paulsen, G. Merz, V. Weichart, xe2x80x9cSolid-Phase Synthesis of O-Glycopeptide Sequencesxe2x80x9d. Angew. Chem. Int. Ed. Engl. 27 (1988); H. Mergler, R. Tanner, J. Gosteli, and P. Grogg. xe2x80x9cPeptide Synthesis by a Combination of Solid-Phase and Solution Methods 1: A New Very Acid-Labile Anchor Group for the Solid-Phase Synthesis of Fully Protected Fragments. Tetrahedron letters 29, 4005 (1988); Merrifield, R. B., xe2x80x9cSolid Phase Peptide Synthesis after 25 Years: The Design and Synthesis of Antagonists of Glucagonxe2x80x9d. Makromol. Chem. Macromol. Symp. 19, 31 (1988)). Furthermore, PCT Patent Application Publication No. WO95/10295 discloses that the amorphous and hygroscopic form of N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl-(L)-xcex2-cyclohexylalanine amide is prepared by sequential synthesis from the C-terminus amino acid as shown in Scheme I. PCT Patent

Application Publication No. WO95/10295 does not disclose the formation of tetra-azacycloalkylalkanoyl peptides and pseudopeptides or, in particular, N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-xcex2-cyclohexylalanine amide from a central di(pseudopeptide or peptide) whereby the N- and C-Terminal ends of the central di(pseudopeptide or peptide) are both coupled with pseudoamino acids and/or aminoacids to form the tetra-azacycloalkyl-alkanoyl peptides and pselidopeptides.
The present invention is directed to a non-hygroscopic stable crystalline form of N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-xcex2-cyclohexyl-alanime amide of formula I. The compound has antithrombotic 
activity, including the inhibition of platelet aggregation and thrombus formation in mammals, and is useful in the prevention and treatment of thrombosis associated with disease states such as myocardial infarction, stroke, peripheral arterial disease and disseminated intravascular coagulation. The invention is also directed to a pharmaceutical composition of the non-hygroscopic stable crystalline form of N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-xcex2-cyclohexylaianine amide and intermediates thereof.
The invention is also directed to processes for preparing a tetra-azacycloalkylalkanoyl peptide or pseudopeptide compound of formula II 
wherein:
A is H;
B is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl, or alkylaralkyl; 
E1 is H;
E2 is the a-carbon side chain of a naturally occuring a-amino acid, H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl, substitued aralkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substituted heterocyclylalkyl, or E1 and E2 taken together with the nitrogen and carbon atoms through which E1 and E2 are linked form a 4-, 5-, 6-, or 7-membered azacycloalkane ring;
G is OR1 or NR1R2;
R1 and R2 are independently H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl, or alkylaralkyl;
R is H, alkyl, aryl, or aralkyl;
m is 1 to 5;
n is 0 to 6; and
p is 1 to 4,
and, in particular, the non-hygroscopic stable crystalline form of N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-xcex2-cyclohexylalanine amide.